Tremendous strides have been taken recently in the molecular biology of taste. As a result, a number of G protein-coupled taste receptors, downstream effectors, and ion channels for taste transduction have been identified. Physiological analyses have not progressed as rapidly, due in part to the difficulty in recording signals from taste cells and taste buds. Additionally, physiological studies are sometimes at odds with molecular biological findings. For example, molecular biological investigations suggest that taste cells express only one class of taste receptors such as bitter receptors or sweet receptors, but not both. However, functional studies show that many taste cells respond to multiple classes of taste chemicals. This and other discrepancies remain unreconciled. We have devised a method that promises to overcome many problems in recording physiological signals in taste cells. We will use calcium microfluorometry, confocal laser microscopy, and slices of mouse lingual epithelium to measure calcium transients elicited by chemical stimuli applied to taste cells. This methodology allows one to apply taste stimuli to the apical chemosensory tips of taste receptor cells and record responses in a semi-intact preparation with high spatial resolution (microns) and good time resolution (100s of msec). We will use this method of calcium imaging to obtain detailed information about signal transduction for stimuli believed to activate G protein-coupled taste receptors, namely sweet, bitter, and umami. We will also investigate taste transduction for acid (sour) stimuli in mouse taste cells. We will correlate physiological responses with expression of candidate effector and target molecules downstream of the taste receptors in identified taste cells by using immunocytochemistry. We will take advantage of genetically altered mice to determine whether physiological responses are missing when specific molecules postulated to be involved in taste transduction are absent. For example, we will test whether calcium transients are elicited by bitter stimuli in taste ceils from gustducin knockout mice. Lastly, we will determine whether single taste receptor cells respond to multiple classes of chemicals or instead are "tuned" to a narrow range of stimuli. Our goal is to bridge the gap between physiological and molecular biological studies and provide a more complete picture of taste transduction mechanisms. [unreadable] [unreadable]